Macroglobulins of Rat Serum

A three-stage method for isolation of a, macroglobulin and a2 macroglobulin from the serum of normal and injured rats is described. The methods successively used, namely gel filtration, ultracentrifugation and chromnatography on DEAE-cellulose, were chosen to minimize loss of tryptic esterase-protecting activity. The two proteins differed slightly with respect to the following properties: mol.wt., a, macroglobulin 7.46 x 10-, a2 macroglobulin 7.16x 105; isoelectric focusing, a., macroglobulin pI 4.4, q2 macroglobulin p14.5. Amino acid analyses were identical, except with respect to tyrosine: a., macroglobulin 3.96±0.24, a2 macroglobulin 3.16+0.32mo1/lOOmol of total amino acids. When isolated from the serum of uninjured rats, a, macroglobulin retained the capacity to bind 1.O5mol of trypsin/mol. However, if isolated from serum 2 days after injury only 0.78mol of trypsin/mol of a, macroglobulin was bound. a,. macroglobulin isolated from this latter serum bound on average 0.97mol of trypsin/mol. When reduced with N-acetylcysteine, both molecules formed subunits of size corresponding to that expected for quarter molecules. When m2 macroglobulin was reduced with dithiothreitol, quarter molecules were again produced. a., macroglobulin, however, when thus treated gave a more complex mixture, containing a component having a mol.wt. of less than 6 x 104. Antisera raised against the two proteins permitted estimation of the concentration of each protein in the plasmas or sera of normal and injured rats. Plasma from normal male rats contained 3.76±0.56mg of acl macroglobulin/ml (n = 33) and 0.016±0.001mg of a2 macroglobulin/mi (n=33). After injury by injection of turpentine and cortisone, the concentrations in plasma were at 3 days 5.19±0.81mg of a, macroglobulin/ml (n = 12) and at 2 days 1.38 ±0.35mg of a2 macroglobulin/ml (n= 12). Antisera to the two proteins did not cross-react with one another. The quarter molecules formed by reduction of both proteins showed increased antigenicity.